Determination of the increased sensitivity of BRCA1-deficient cells to poly (ADP-ribose) polymerase (PARP) inhibitors resulted in development of effective and targeted cancer therapies. Approval of PARP inhibitors as mono- or combination- therapies for breast and ovarian cancers demonstrate the importance of identifying specific genes and pathways that mediate pathway alterations in cancer cells. With about 450 proteins participating in double-stranded damage repair (DDR) pathway, it is expected that some of the synergistic interactions between DDR genes are yet to be discovered. Systematic approaches with large datasets will accelerate discovery of such interactions by providing functional insights into formation of testable molecular models. The aim of the proposed research plan is to discover new factors involved in DDR and cancer progression by complementing a genetic-chemical interaction study with mechanistic and structural characterization in an orthogonal manner. Specific Aims of this proposal are designed to independently reflect the collaborative expertise of the Krogan lab in deducing biological insights from collection and analysis of large datasets, while taking advantage of the resources available in UCSF. The goal of Aim1 is to identify BRCA1 interactors that are functionally important for tumor suppression using a CRISPR-Cas9 based screen. The differential effects of knocking out around 92 BRCA1-interacting genes, including novel interactors that were identified in preliminary proteomics studies, on sensitivity to different kinds of chemotherapy drugs will be evaluated. This screen will be followed by more targeted drug treatments in order discern proteins that form complexes. The targeted gene selection approach that will be employed here has the advantage of enriching for proteins that are likely to have related functions. Similar to genetic interaction screens, proteins that are involved in the same protein complexes are expected to show sensitivity to same kinds of drugs. Aim2 will establish the role of neddylation and the implications of the GPS1-BRCA1 complex on homologous recombination. The role of neddylation in homologous recombination is understudied and results have been contradictory. Preliminary experiments point to a previously unexplored interaction between GPS1, a COP9-signalosome subunit, and BRCA1. Quantitative and targeted mass spectrometry-based analysis, along with biochemical assays will be used to study the role of neddylation and COP9 signalosome on BRCA1 levels in the cell. Aim3 will determine the cryo-EM structure of prevailing BRCA1-complexes. This aim will be completed in collaboration with the Agard Lab, using a derivatized affinity grid technology. Determination of the structure of BRCA1-complexes will provide a mechanistic overview of the interactions, as well as enable targeted drug engineering. Successful execution of this project will lead to the discovery of new therapeutic targets while expanding the number of patients that could benefit from already FDA-approved agents.